The heme-containing enzyme commonly known as chlorite dismutase (Cld) catalyzes the unimolecular decomposition of chlorite ion, ClO2-, as illustrated in Scheme 1. The reaction is thought to proceed via formation of compound I (2) through a heme-catalyzed isomerization of O-bound chlorite, FeIIIClO2-, to coordinated peroxyhypochlorite, FeIII-OOCl- (3), followed by an intramolecular redox reaction that releases O2, and Cl-. Recent spectroscopic and kinetic work has led to the suggestion that the catalytic mechanism proceeds through a two-electron oxidized heme intermediate having properties characteristic of compound I (Cpd-I). Its formation is thought to occur with release of the two-electron reduced fragment, hypochlorite, which recombines with the yl-oxygen atom of Cpd-I to yield a peroxyhypochlorite complex. This complex is unstable with respect to release of O2 and Cl-. Reactions that evolve O2 are rare in biology, with that catalyzed by the O2-evolving tetranuclear Mn cluster of PSII being the only other known example. The precise nature of the Cld intermediates and the modulation of their reactivities by the enzyme remain to be elucidated. The overarching goal of this work is to clarify the structural basis for the spectacular catalytic efficiency and mechanistic specificity of this unique O2-evolving reaction. The specific aims focus on spectroscopic (primarily resonance Raman, rR) and kinetic (freeze-quench) approaches to determining atom connectivities, structures and electronic properties of intermediates in the catalytic decomposition of ClO2- by Cld from Dechloromonas aromatica RCB. Specifically, this proposed project aims to: 1. further clarify the roles of distal pocket residues in directing substrate binding and Cl-O bond scission to yield Cpd-I. 2. probe the nonbonded interactions between the ferryl moiety of the Cpd-I intermediate and the distal pocket in the freeze quenched reaction of ferric Cld with peracetic acid by resonance Raman spectroscopy. 3. probe structure and bonding in the first catalytic intermediate in the reaction between ferric Cld and the substrate, ClO2-. 4. investigate the structural and electronic properties of the rebounded state. PUBLIC HEALTH RELEVANCE: The goal of this project is to build a basic understanding of how biological organisms make the oxygen that we breath. This knowledge will provide the foundation for development of medical and materials technologies for specialized oxygen delivery systems. It can also be applied to the development of systems for removing toxic chlorine-based pollutants from waste and ground water.